日本血吸虫Tsunagi样蛋白编码基因功能的鉴定
摘要
血吸虫病是严重危害人民身体健康,阻碍社会经济发展的重大传染病。日本血吸虫致病主要是由于虫卵在肝脏大量沉积形成的虫卵肉芽肿及其纤维化。控制血吸虫性别分化、性成熟及雌虫产卵成为防治血吸虫病的重要策略之一。
为了解日本血吸虫Tsunagi蛋白的功能,我们用RNAi技术下调Tsunagi基因的转录水平,再观察干扰作用后虫体的发育(尤其是生殖系统发育)是否与正常虫体之间存在差异。按照双链RNA(dsRNA)体外合成试剂盒要求,我们从日本血吸虫童虫cDNA文库中扩增出Tsunagi基因,将其片段正、反义链连接上T7启动子,然后把连接有T7启动子的目的基因的正、反义链进行PCR扩增。将PCR产物转录成单链RNA(ssRNA)并将其纯化,最后把一对ssRNA混合合成dsRNA。阴性对照组我们采用试剂盒中提供的基因。在BTX电穿孔仪上,设定参数为125V电压、20ms脉冲时值、1次脉冲次数,将dsRNA电穿孔转染入机械制备的日本血吸虫童虫体内,体外培养于电转后第1, 3, 5天收集童虫,按TRIzol方法同时提取童虫的总RNA及总蛋白。经实时荧光定量PCR及Western blotting分别检测Tsunagi基因和蛋白表达水平变化。结果发现Tsunagi基因的转录水平实验组分别于电穿孔后第1, 3, 5天较阴性对照组下降16%,57%,75%。该基因的蛋白水平在第1, 3, 5天较阴性对照组下降15%,38%,52%。实验结果证明dsRNA可以特异性的抑制日本血吸虫靶基因以及蛋白的表达且效果明显。经dsRNA电转的童虫注射入小鼠体内,6周后取出虫体通过一系列处理制成标本,在激光共聚焦显微镜下观察虫体内部各器官形态特征及测量虫体体长、体宽、睾丸长、睾丸宽、睾丸面积、卵巢长、卵巢宽、卵巢面积。结果发现SjTsunagi dsRNA组中8条中3～4条雄虫睾丸内有大量精子出现,而雌虫中卵巢,卵黄腺中却没有明显特征变化;测得各项指标经SPSS 13.0统计软件分析阴性对照组与SjTsunagi dsRNA组的体宽、睾丸长、睾丸宽、睾丸面积、卵巢长、卵巢宽、卵巢面积均有显著性差异。由此结果我们得出Tsunagi基因在日本血吸虫中是生殖相关基因,在生殖系统器官的正常发育起一定作用。
Schistosomiasis is still a serious infectious disease which is harmful to people’s health and obstruct the social and economic development. The schistosome eggs are retained in the liver of the final host where they elicit inflammatory immune responses, which lead to formation of the granuloma and fibrosis, the major pathological effects of schistosomiasis. Controlling sexual dimorphism, sexual maturation and labour division may be effective in prevention of schistosomiasis.
According to in vitro dsRNA synthesis kit demand, we amplified Tsunagi gene from schistosomulum cDNA library. Then we amplified the positive and anti-sense strand with promoter T7 by PCR then transcript into ssRNA and purification, finally syntheses dsRNA with a couple of ssRNA.We use the gene provided by the kit in control group. We electroporated with dsRNA to schistosomulum at 125V for 20ms,1 pulse using an Electro Square PoratorTM ECM830(BTX).Aliquots of parasites were harvested respectively in day 1,3,5 after electroporation.Total RNA,DNA and proteins were isolated using TRIZOL Reagent according to the manufacturer's guidelines. Levels of Tsunagi mRNA and proteins were determined by RT-PCR and Western blotting analysis. The results showed that SjTsunagi mRNA levels were decreased by16%,57%,75% in test group,respectively,at day 1,3,5 after electroporation.The SjTsunagi protein expression levels were decreased by15%,38%,52% in test group,respectively,at day 1,3,5 after electroporation.The results suggested the dsRNA could inhibit the expression of the target gene and the protein specifically and efficiently. The schistosomula which were electroporated with dsRNA were injected into the mice,and we made them into specimen through a procession of methods after 6 weeks,then oberserved the morphology characteristics of each organ and measured the length and width of the worms,the length,width and area of the testicular lobes,the length,width and area of the ovaries under the confocal laser scanning microscopy.The results show there are many spermatozoa in testicular lobes of three to four worms of eight male in SjTsunagi group and no changes in ovary and vitelline gland of female. The adult worms which were infected 6 weeks in SjTsunagi dsRNA group have significant differences from control group in width of the worms, the length, width and area of the testicular lobes, the length, width and area of the ovaries through SPSS13.0 statistics software package. So we concluded that Tsunagi is the gene associated with reproduction in schistosomiasis japonicum.
为了解日本血吸虫Tsunagi蛋白的功能,我们用RNAi技术下调Tsunagi基因的转录水平,再观察干扰作用后虫体的发育(尤其是生殖系统发育)是否与正常虫体之间存在差异。按照双链RNA(dsRNA)体外合成试剂盒要求,我们从日本血吸虫童虫cDNA文库中扩增出Tsunagi基因,将其片段正、反义链连接上T7启动子,然后把连接有T7启动子的目的基因的正、反义链进行PCR扩增。将PCR产物转录成单链RNA(ssRNA)并将其纯化,最后把一对ssRNA混合合成dsRNA。阴性对照组我们采用试剂盒中提供的基因。在BTX电穿孔仪上,设定参数为125V电压、20ms脉冲时值、1次脉冲次数,将dsRNA电穿孔转染入机械制备的日本血吸虫童虫体内,体外培养于电转后第1, 3, 5天收集童虫,按TRIzol方法同时提取童虫的总RNA及总蛋白。经实时荧光定量PCR及Western blotting分别检测Tsunagi基因和蛋白表达水平变化。结果发现Tsunagi基因的转录水平实验组分别于电穿孔后第1, 3, 5天较阴性对照组下降16%,57%,75%。该基因的蛋白水平在第1, 3, 5天较阴性对照组下降15%,38%,52%。实验结果证明dsRNA可以特异性的抑制日本血吸虫靶基因以及蛋白的表达且效果明显。经dsRNA电转的童虫注射入小鼠体内,6周后取出虫体通过一系列处理制成标本,在激光共聚焦显微镜下观察虫体内部各器官形态特征及测量虫体体长、体宽、睾丸长、睾丸宽、睾丸面积、卵巢长、卵巢宽、卵巢面积。结果发现SjTsunagi dsRNA组中8条中3～4条雄虫睾丸内有大量精子出现,而雌虫中卵巢,卵黄腺中却没有明显特征变化;测得各项指标经SPSS 13.0统计软件分析阴性对照组与SjTsunagi dsRNA组的体宽、睾丸长、睾丸宽、睾丸面积、卵巢长、卵巢宽、卵巢面积均有显著性差异。由此结果我们得出Tsunagi基因在日本血吸虫中是生殖相关基因,在生殖系统器官的正常发育起一定作用。
Schistosomiasis is still a serious infectious disease which is harmful to people’s health and obstruct the social and economic development. The schistosome eggs are retained in the liver of the final host where they elicit inflammatory immune responses, which lead to formation of the granuloma and fibrosis, the major pathological effects of schistosomiasis. Controlling sexual dimorphism, sexual maturation and labour division may be effective in prevention of schistosomiasis.
According to in vitro dsRNA synthesis kit demand, we amplified Tsunagi gene from schistosomulum cDNA library. Then we amplified the positive and anti-sense strand with promoter T7 by PCR then transcript into ssRNA and purification, finally syntheses dsRNA with a couple of ssRNA.We use the gene provided by the kit in control group. We electroporated with dsRNA to schistosomulum at 125V for 20ms,1 pulse using an Electro Square PoratorTM ECM830(BTX).Aliquots of parasites were harvested respectively in day 1,3,5 after electroporation.Total RNA,DNA and proteins were isolated using TRIZOL Reagent according to the manufacturer's guidelines. Levels of Tsunagi mRNA and proteins were determined by RT-PCR and Western blotting analysis. The results showed that SjTsunagi mRNA levels were decreased by16%,57%,75% in test group,respectively,at day 1,3,5 after electroporation.The SjTsunagi protein expression levels were decreased by15%,38%,52% in test group,respectively,at day 1,3,5 after electroporation.The results suggested the dsRNA could inhibit the expression of the target gene and the protein specifically and efficiently. The schistosomula which were electroporated with dsRNA were injected into the mice,and we made them into specimen through a procession of methods after 6 weeks,then oberserved the morphology characteristics of each organ and measured the length and width of the worms,the length,width and area of the testicular lobes,the length,width and area of the ovaries under the confocal laser scanning microscopy.The results show there are many spermatozoa in testicular lobes of three to four worms of eight male in SjTsunagi group and no changes in ovary and vitelline gland of female. The adult worms which were infected 6 weeks in SjTsunagi dsRNA group have significant differences from control group in width of the worms, the length, width and area of the testicular lobes, the length, width and area of the ovaries through SPSS13.0 statistics software package. So we concluded that Tsunagi is the gene associated with reproduction in schistosomiasis japonicum.
引文
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9.王晓楠,雷黎,赵志荣.日本血吸虫Tsunagi蛋白基因的获得、表达及初步鉴定.中国人兽共患病学报, 2008, 24(2): 120-124.
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2. Basch PF. Cultivation of Schistosoma mansoni in vitro.Ⅱ. Production of infertile eggs by worm pairs cultured from cercariae. J Parasitol, 1981, 67: 186-189.
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6. Kataoka N, Yong J, Kim VN, et al. Pre-mRNA splicing imprints mRNA in the nucleus with a novel RNA-binding protein that persist in the cytoplasm. Mol Cell, 2000, 6: 673-682.
7. Mohr SE, Dillon ST, Boswell RE. The RNA-binding protein Tsunagi interacts with Mago nashi to establish polarity and localize oskar mRNA during Drosophila oogenesis. Genes Dev, 2001, 15: 2886-2899.
8. Kawano T, Kataoka N, Dreyfuss G, et al. Ce-Y14 and MAG-1, components of the exon-exon junction complex, are required for embryogenesis and germline sexual switching in Caenorhabditis elegans. Machanisms of Development, 2004, 121: 27-35.
9.王晓楠,雷黎,赵志荣.日本血吸虫Tsunagi蛋白基因的获得、表达及初步鉴定.中国人兽共患病学报, 2008, 24(2): 120-124.
10. Fire A, Xu S, Montgomery MK, et, al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, 1998, 391: 806-811.
11. Zhang W, Yang H, Kong X, et al. Inhibition of respiratory syncytial virus infection with intranasal siRNA nanoparticles targeting the viral NS1 gene. Nat Med, 2005, 11(1):56-62.
12. Fowler T, Bamberg S, Moller P, et al. Inhibition of Marburg virus proteinexpression and viral release by RNA interference. J Gen Virol, 2005, 86:1181-1188.
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15. Timmons L, Fire A. Specific interference by ingested dsRNA. Nature, 1998, 395(6705): 854.
16. Correnti JM, Pearce EJ. Transgene expression in Schistosoma mansoni: introduction of RNA into schistosomula by electroporation. Molecular and Biochemical Parasitology, 2004, 137: 75–79.
17. Boswell RE, Prout ME, Steichen JC. Mutations in a newly identified Drosophila melanogaster gene, mago nashi, disrupt germ cell formation and result in the formation of mirror-image symmetrical double abdomen embryos. Development, 1991, 113: 373-384.
18. Ephrussi A, Dickinson LK, Lehnmann R. Oskar organizes the germ plasm and directs localization of the posterior determinant nanos. Cell, 1991, 66: 37-50.
19. Kim-Ha J, Smith JL, Macdonald PM. Oskar mRNA is localized to the posterior pole of the Drosophila oocyte. Cell, 1991, 66: 23-35.
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